本文關(guān)鍵詞：多種材料中紫杉醇合成能力的基因證據(jù)探索　出處：《中國(guó)林業(yè)科學(xué)研究院》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 紫杉醇 紫杉二烯合酶 宏基因組 原核表達(dá)

【摘要】：紫杉醇(Taxol)是一種廣泛應(yīng)用于治療肺癌、卵巢癌和乳腺癌的復(fù)雜二萜化合物,它首先從短葉紅豆杉(Taxus brevifolia)樹(shù)皮中分離得到。現(xiàn)在紫杉醇主要來(lái)源為生長(zhǎng)緩慢的紅豆杉樹(shù)木,紫杉醇的供應(yīng)尚嚴(yán)重不足。因此,尋找紫杉醇的替代來(lái)源非常重要。在過(guò)去的20多年里,人們對(duì)一些生產(chǎn)紫杉醇的替代方法進(jìn)行了探索,其中包括產(chǎn)紫杉醇微生物的研究。人們?cè)诜蛛x具有紫杉醇合成能力的內(nèi)生菌(真菌和細(xì)菌)時(shí),通常使用可培養(yǎng)的方法,即通過(guò)可培養(yǎng)的方法在培養(yǎng)基上分離可培養(yǎng)菌,但是,這種方法不能分離不可培養(yǎng)的內(nèi)生菌,造成對(duì)菌株的篩選不全面,可能會(huì)錯(cuò)過(guò)高產(chǎn)的菌株。下一代測(cè)序技術(shù)中的宏基因組測(cè)序已經(jīng)應(yīng)用到對(duì)有些土壤微生物群落的物種和基因組成研究。但到目前為止,仍沒(méi)有關(guān)于紅豆杉根部相關(guān)微生物的菌種鑒定和基因組成進(jìn)行研究的報(bào)道。在本研究中,我們使用可以靶定16S和18S r DNA的引物,利用bar-coded高通量測(cè)序技術(shù),對(duì)紅豆杉根部相關(guān)的細(xì)菌和真菌做了一個(gè)全面的調(diào)查。通過(guò)宏基因組研究調(diào)查了紅豆杉根部相關(guān)微生物的物種分布和基因組成,預(yù)測(cè)了紅豆杉根部相關(guān)微生物中和紫杉醇合成相關(guān)的基因,旨在通過(guò)對(duì)這些基因的研究來(lái)評(píng)估紅豆杉根部相關(guān)微生物中是否存在具有紫杉醇合成能力的菌株。除產(chǎn)紫杉醇內(nèi)生真菌以外,人們還在甜菜樹(shù)(Yunnanopilia longistaminata)、粗榧(Cephalotaxus sinensis)、榛子(Corylus heterophylla Fisch)、馬尾松(Pinus massoniana Lamb)、羅漢松(Podocarpus macrophyllus)等植物中發(fā)現(xiàn)了包括紫杉醇在內(nèi)的多種紫杉烷類化合物的存在,但尚沒(méi)有足夠的證據(jù)證明其具有紫杉烷類化合物合成的能力。眾所周知,證明紫杉二烯合酶(Taxadiene synthease,TS)基因的存在是證明這種能力存在的最好證據(jù)。所以,本研究對(duì)產(chǎn)紫杉醇內(nèi)生真菌Penicillium aurantiogriseum NRRL 62431、甜菜樹(shù)及粗榧的基因組數(shù)據(jù)或轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行了TS基因的預(yù)測(cè),并對(duì)預(yù)測(cè)的類紫杉二烯合酶基因(Taxadiene synthease like gene,TSL)進(jìn)行了功能鑒定,試圖從分子水平上找到其具有合成紫杉醇能力的證據(jù)。通過(guò)實(shí)驗(yàn)發(fā)現(xiàn),我們篩選的基因在體外都不具備紫杉二烯合酶的功能,只有粗榧中的Cs TSL能夠催化GGPP合成紫杉烷類化合物的中間產(chǎn)物verticiol。本文主要研究結(jié)果如下:1.對(duì)南方紅豆杉(Taxus chinensis(Pilger)Rehd)根部進(jìn)行了微生物多樣性的調(diào)查。通過(guò)16S r DNA測(cè)序共鑒定出640個(gè)細(xì)菌OTU(Operational Taxonomic Units)。本研究發(fā)現(xiàn)變形菌(Proteobacteria)是南方紅豆杉根部最豐富的細(xì)菌種類。遺憾的是并沒(méi)有找到任何報(bào)道過(guò)的能夠合成紫杉醇的細(xì)菌物種。之后通過(guò)18S r DNA測(cè)序共鑒定出187個(gè)真菌OTU。發(fā)現(xiàn)擔(dān)子菌門(Basidiomycota)是南方紅豆杉根部最豐富的真菌種類。我們發(fā)現(xiàn)絲齒菌屬(Hyphodontia)、Hemimycena屬和Phialocephala屬是三個(gè)南方紅豆杉根部真菌優(yōu)勢(shì)屬,這也是首次在所有紅豆杉屬中報(bào)道發(fā)現(xiàn)這3個(gè)屬。本文中發(fā)現(xiàn)了187個(gè)真菌的OTU,這比以往報(bào)道的任何依靠培養(yǎng)的方法發(fā)現(xiàn)的菌株都要多,這樣說(shuō)明南方紅豆杉根部寄生著多樣的真菌。對(duì)南方紅豆杉根部進(jìn)行了宏基因組調(diào)查,經(jīng)分析發(fā)現(xiàn)Contig主要分布在34個(gè)細(xì)菌的門中和9個(gè)真菌的門中。甲型變形菌(Alphaproteobacteria)、丙型變形菌(Gammaproteobacteria)和芽孢桿菌(Bacilli)是最占優(yōu)勢(shì)的細(xì)菌類群,而酵母菌(Saccharomycetes)、球囊菌綱(Glomeromycetes)和糞殼菌綱(Sordariomycetes)是最占優(yōu)勢(shì)的真菌類群。并發(fā)現(xiàn)可能存在5個(gè)具有紫杉醇合成能力的微生物物種。2.對(duì)一個(gè)產(chǎn)紫杉醇內(nèi)生真菌P.aurantiogriseum NRRL 62431的基因組數(shù)據(jù)進(jìn)行了分析,預(yù)測(cè)到一個(gè)紫杉二烯合酶和一個(gè)GGPP合酶。首先完整克隆出內(nèi)生真菌P.aurantiogriseum NRRL 62431的一個(gè)TSL基因的開(kāi)放閱讀框(Open Reading Frame,ORF),并研究了其活性,發(fā)現(xiàn)其反應(yīng)產(chǎn)物中并沒(méi)有紫杉二烯,說(shuō)明這個(gè)TSL基因可能不是紫杉二烯合酶基因。當(dāng)然,也可能是該P(yáng)a TSL基因在體外表達(dá)的蛋白不具備紫杉二烯合酶的功能。同時(shí)完整克隆出內(nèi)生真菌P.aurantiogriseum NRRL 62431的GGPPS基因的ORF序列,并用生物信息學(xué)預(yù)測(cè)和分析了Pa GGPPS的氨基酸序列、理化性質(zhì)、疏水性/親水性、功能結(jié)構(gòu)域和二級(jí)結(jié)構(gòu)。該基因在大腸桿菌成功進(jìn)行了表達(dá),產(chǎn)生了可溶性的蛋白,并通過(guò)試劑盒進(jìn)行了純化。這些工作為下一步深入研究該內(nèi)生真菌的紫杉醇合成途徑中此基因的作用和構(gòu)建高產(chǎn)紫杉醇基因工程菌株提供了一定的基礎(chǔ)。3.對(duì)含有紫杉烷類化合物的甜菜樹(shù)(Y.longistaminata)進(jìn)行了一系列研究。首先對(duì)其進(jìn)行了轉(zhuǎn)錄組測(cè)序。通過(guò)轉(zhuǎn)錄組測(cè)序,總共得到了約4.8G的序列數(shù)據(jù),組裝得到了51,744條Unigene序列。接著,對(duì)轉(zhuǎn)錄組進(jìn)行了KEGG代謝途徑注釋,共有6,513個(gè)Unigene得到注釋,這些轉(zhuǎn)錄本主要被注釋到了335條代謝途徑中,其中,有41條Unigene被注釋到萜類合成中,這些數(shù)據(jù)中可能含有甜菜樹(shù)能夠合成紫杉烷類化合物的基因證據(jù)。通過(guò)對(duì)轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行本地Blast篩選,共發(fā)現(xiàn)2個(gè)TSL基因片段(Yl TSL1和Yl TSL2)。然后針對(duì)Yl TSL1的功能展開(kāi)了研究,利用RACE-PCR技術(shù)從甜菜樹(shù)中克隆得到了Yl TSL1基因的c DNA全長(zhǎng),并研究了其蛋白活性,發(fā)現(xiàn)其并不具備二萜合酶的功能,但是卻有α-法尼烯合酶的功能,所以甜菜樹(shù)中得到的基因Yl TSL1并非紫杉二烯合酶基因,可以確定為α-法尼烯合酶基因。隨后在展開(kāi)了對(duì)Yl TSL2基因的研究,利用RACE-PCR技術(shù)從甜菜樹(shù)中克隆得到了Yl TSL2基因的c DNA全長(zhǎng),并研究了其蛋白活性,發(fā)現(xiàn)Yl TSL2基因?yàn)樨悮ど枷┖厦富颉?.對(duì)粗榧(C.sinensis)中的TSL基因進(jìn)行了研究。通過(guò)分析Gen Bank中已有柱冠粗榧(C.harringtonia)的轉(zhuǎn)錄組數(shù)據(jù)(SRA:SRR064395),預(yù)測(cè)到一個(gè)TSL基因(Cs TSL),通過(guò)RACE-PCR從粗榧(C.sinensis)中克隆到了Cs TSL基因的c DNA全長(zhǎng),并進(jìn)行了其蛋白活性的研究,發(fā)現(xiàn)其在體外并不具備紫杉二烯合酶的功能,但是可以催化GGPP合成verticiol和17-methyl-5a-androst-2-ene兩種化合物,后者為反應(yīng)的主產(chǎn)物,是一種激素類似物。而對(duì)于副產(chǎn)物verticiol,Erdtman等曾推測(cè)其是紫杉二烯合成過(guò)程中的中間化合物。Cs TSL為何不能直接催化GGPP形成紫杉二烯?可能是因其沒(méi)有催化最后一步環(huán)化的活性中心,這需要在后續(xù)研究中進(jìn)一步分析。
[Abstract]:Paclitaxel (Taxol) is a widely used in the treatment of lung cancer, two complex triterpenoids, ovarian cancer and breast cancer, it first from Taxus brevifolia (Taxus brevifolia) isolated from the bark. Now paclitaxel is mainly from the slow growth of Taxus trees, taxol supply is seriously insufficient. Therefore, looking for paclitaxel an alternative source of alcohol is very important. In the past 20 years, the people of some alternative methods of taxol production are explored, including the study of taxol producing microorganisms. It has the ability of taxol biosynthesis in isolated endophytic fungi (fungi and bacteria), usually using the method of culture, namely through methods cultured on the culture medium separation of culturable bacteria, however, this method cannot be separated from the non culturable endophytic bacteria, resulting in screening of strains is not comprehensive, may miss the high-yield strains. The next generation sequencing technology Macro genome sequencing has been applied to species and genes on some soil microbial community composition research. But so far, the identification and gene is still no microbial composition of Taxus roots on research reports. In this study, we can use primers targeting 16S and 18S R DNA, using Qualcomm bar-coded the amount of sequencing of Taxus root associated bacteria and fungi has done a comprehensive survey. The species distribution and composition of microbial gene of Taxus roots were investigated through metagenomic research, prediction of yew root microorganism and taxol synthesis related genes, to assess whether there has the ability of synthesis of taxol strains the root of Taxus microorganisms through the study of these related genes. In addition to Taxol producing endophytic fungi, people still beet tree (Yunnanop Ilia LONGISTAMINATA) (Cephalotaxus sinensis), Cephalotaxus sinensis (Corylus heterophylla Fisch), hazelnut, masson pine (Pinus massoniana Lamb) (Podocarpus macrophyllus), Podocarpus found a variety of taxanes including taxol in the presence of other plants, but there is not enough evidence to prove its ability of taxanes biosynthesis. As everyone knows, that of taxol diene synthase (Taxadiene, synthease, TS) gene is the best evidence of this ability exist. Therefore, the study of taxol producing endophytic fungi Penicillium aurantiogriseum NRRL 62431, genome or transcriptome data beet tree and prediction of Cephalotaxus sinensis TS gene, and to predict the taxane diene synthase gene (Taxadiene synthease like gene, TSL) of the functional analysis, trying to find the synthesis of taxol from the molecular level The ability of evidence. Through the experiment, we screened the gene does not have the taxadiene synthase function in vitro, Cs TSL can only in Cephalotaxus intermediate verticiol. catalyzed GGPP the main results of this paper are as follows: 1.. Synthesis of taxane compounds of Taxus chinensis (Taxus chinensis (Pilger) Rehd) conducted a survey of microbial diversity in root the 16S R DNA. By sequencing identified 640 bacteria OTU (Operational Taxonomic Units). The study found that the deformation of bacteria (Proteobacteria) is the most abundant species of Taxus chinensis roots. Unfortunately did not find any reported to bacterial species on the biosynthesis of taxol. After 18S R DNA sequencing Co OTU. found that identified 187 fungi Basidiomycota (Basidiomycota) is the most abundant fungal species of Taxus chinensis root. We found that hyphodontia (Hyphodontia, Hemim) Ycena and Phialocephala genus are three Taxus root fungi genera, this is the first time in all reported in Taxus found these 3 genera. Found 187 fungi OTU in this paper, the methods of cultivating it rely on any than previously reported strains are more, this shows that Taxus chinensis root parasitic a variety of fungi. The metagenomic survey of Taxus roots, the analysis shows that Contig mainly distributed in the 34 bacteria and 9 fungi door door. Alpha Proteobacteria (Alphaproteobacteria), C Proteobacteria (Gammaproteobacteria) and Bacillus (Bacilli) is the most dominant bacteria, and yeast (Saccharomycetes), glomeromycetes (Glomeromycetes) and sordariomycetes (Sordariomycetes) is the most dominant fungal taxa. And found that there are 5 has the ability of taxol biosynthesis in microorganisms Species.2. analysis of the genomic data of a taxol producing endophytic fungus P.aurantiogriseum NRRL 62431, predict a taxadiene synthase and GGPP synthase TSL gene. A first complete clone of endophytic fungus P.aurantiogriseum NRRL 62431 open reading frames (Open, Reading, Frame, and ORF) study on the activity, found that the reaction product is not taxadiene, this TSL gene may not taxadiene synthase gene. Of course, it may be the expression of Pa TSL gene in vitro protein does not have the taxadiene synthase function. The ORF sequence of GGPPS gene and cloned complete P.aurantiogriseum endophytic fungi NRRL 62431 the use of bioinformatics prediction and analysis of the amino acid sequence of Pa GGPPS, physicochemical properties, hydrophobicity / hydrophilicity, functional domains and two level structure. The gene in Escherichia coli The expression of the soluble protein, and the kit was purified. This work provides a basis for a certain.3. of the beet tree containing taxanes taxol biosynthesis pathway of this gene further study the endophytic fungi and the role of constructing high-yield paclitaxel gene engineering strain (Y.longistaminata) conducted a series of studies. Firstly, transcriptome sequencing. By transcriptome sequencing, a total sequence of data about 4.8G, assembled by 51744 Unigene sequences. Then, on the transcriptome metabolic pathways of KEGG notes, a total of 6513 Unigene notes, these major transcripts were annotated the 335 pathways, among them, 41 Unigene were annotated to terpenoid synthesis, may contain beet gene trees can evidence synthesis of taxanes through these data. Local Blast screening of transcriptome data, 2 were found in TSL gene fragments (Yl TSL1 and Yl TSL2). Then studied the function of TSL1 in Yl, cloned from beet tree obtained the full-length Yl gene of TSL1 C DNA by RACE-PCR technology, and studied its protein activity, it does not have two terpene synthase function, but alpha farnesene synthase function, so the Yl TSL1 gene of beet tree is not the taxadiene synthase gene can be identified as the alpha farnesene synthase gene. Then carries out the research on Yl TSL2 gene, cloned from sugar beet tree has been the full-length Yl TSL2 gene C DNA by using RACE-PCR technology, and studied its protein activity, found that Yl TSL2 gene for kaurene synthase gene.4. of Cephalotaxus sinensis (C.sinensis) in the TSL gene were studied. Through the analysis of the existing Bank Gen column (C.harringtonia) to the crown of Cephalotaxus sinensis Recorded data group (SRA:SRR064395), predicted that a TSL gene (Cs TSL), RACE-PCR (C.sinensis) from Cephalotaxus sinensis by cloning the full-length Cs gene of TSL C DNA, and studied its protein activity, found that it does not have the taxadiene synthase function in vitro, but can catalyze the synthesis of GGPP verticiol and 17-methyl-5a-androst-2-ene two compounds, the latter is the main reaction products, is a kind of hormone analogues. The by-products such as Erdtman verticiol, have speculated that it is why taxadiene synthesis process of the intermediate compound.Cs TSL directly catalyzed GGPP formation of taxadiene? Probably because it is not the last step of catalytic cyclization the activity center, which need further analysis in the follow-up study.

【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q933;Q943.2

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2 王偉,石青,朱平,歐陽(yáng)濤,李y，

本文編號(hào)：1436999